Chemical process for preparing amide derivatives of antibiotic A 40926

ABSTRACT

Improved chemical process for preparing the compounds of formula (I), wherein: R 1  represents (C 9 -C 12 )alkyl; M represents hydrogen, α-D-mannopyranosyl or 6-O-acetyl-α-D-mannopyranosyl and Y represents an amino group of formula —NR 2 -alk 1 -(NR 3 -alk 2 ) p -(NR 4 -alk 3 ) q -W. One aspect of the invention refers to the preparation of the intermediate compound of formula (III) (which corresponds to the above compound of formula (I)                    
     wherein R 1  and M are as above defined and Y is hydroxy), by reacting a compound of formula (II) (which corresponds to the above compound of formula (I) wherein R 1  and M are as above defined and Y is hydroxymethyl), with a (C 1 -C 4 )alkanol in the presence of a concentrated mineral acid, using the same alkanol as the reaction solvent and submitting the obtained ester compound to a reductive process by adding an alkali metal borohndride into the same reaction mixture. A further aspect of the invention refers to the amidation reaction for obtaining the compound of formula (I), which is carried out by reacting a compound of formula (III) with a suitable amine of formula (IV): NHR 3 -alk 1 -(NR 4 -alk 2 ) p -(NR 5 -alk 3 ) q -W, wherein R 3 , R 4 , R 5 , alk 1 , alk 2 , alk 3 , p, q and W are as defined in formula (I), in an inert organic solvent, in the presence of a condensing agent and setting the initial pH of the mixture (measured after diluting a sample of the reaction mixture with 9 volumes of water) at a value of from 6.5 to 9.0. By combining the above improved steps into a single process, it is possible to set up a particularly convenient process for preparing the compounds of formula (I). In addition, a purification method involving the use of polyamide resins and aqueous eluents is provided.

The present invention refers to an improved chemical process forpreparing amide derivatives of antibiotic A 40926 of formula I:

wherein:

R₁ represents (C₉-C₁₂)alkyl;

M represents hydrogen, α-D-mannopyranosyl or6-O-acetyl-α-D-mannopyranosyl;

Y represents an amino group of formula

—NR₂-alk₁-(NR₃-alk₂)_(p)-(NR₄-alk₃)_(q)-W

wherein:

R₂ represents hydrogen or (C₁-C₄)alkyl;

alk₁, alk₂

and alk₃ each independently represent a linear or branched alkylene of 2to 10 carbon atoms;

p and q are integers which independently represent zero or 1;

R₃ and R₄ each independently represent hydrogen, (C₁-C₄)alkyl or

R₂ and R₃ taken together represent a (C₂-C₄)alkylene moiety connectingthe two nitrogen atoms with the proviso that p is 1; or

R₃ and R₄ taken together represent a (C₂-C₄)alkylene moiety connectingthe two nitrogen atoms with the proviso that both p and q are 1;

W represents hydrogen, (C₁-C₄)alkyl, amino, (C₁-C₄)alkylamino,di(C₁-C₄)alkylamino, amino substituted with one or twoamino(C₂-C₄)alkylene moieties or with one or two(C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties or with one or twodi(C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties, or, when both p and q arezero, taken together with the moiety —NR₃-alk₁- it may also representpiperazino or 4-methylpiperazino.

Antibiotic A 40926 is a glycopeptide antibiotic complex which has beenisolated from a culture of Actinomadura, named Actinomadura sp. ATCC39727, in a culture medium containing assimilable sources of carbon,nitrogen, and inorganic salts (see U.S. Pat. No. 4,935,238). Accordingto the procedure described in the above cited patent the recovery of theantibiotic complex, whose major factors have been named factor A, factorB, factor B₀, factor B₁, factor PA, and factor PB, includes submittingthe fermentation broths, after filtration or after a preliminarypurification, to affinity chromatography on immobilizedD-alanyl-D-alanine.

The A 40926 factors so far identified can be represented by formula (II)below wherein R is carboxy, R₁ represents a (C₉-C₁₂)alkyl group, and Mrepresents an α-D-mannopyranosyl or a 6-O-acetyl-α-D-mannopyranosylgroup.

More particularly, antibiotic A 40926 factor A is a compound of theabove formula (II) wherein R is carboxy, R₁ represents n-decyl, and Mrepresents α-D-mannopyranosyl. According to the most recent studies, thesubstance identified in the above mentioned EP-177882 as antibiotic A40926 factor B, actually consists of two closely related components.Antibiotic A 40926 factor B₀ is indeed the main component of factor B,and corresponds to the compound of the above formula (II) wherein R iscarboxy, R₁ represents 9-methyldecyl, and M representsα-D-mannopyranosyl.

The minor component of factor B is named factor B₁ and differs fromfactor B₀ only in that R₁ represents n-undecyl (E. Riva et al,Chromatographia, Vol. 24, 295, 1987).

Antibiotic A 40926 factor PA and factor PB differ from the correspondingfactor A and B in that the mannose unit is replaced by a6-O-acetyl-α-D-manno-pyranose unit.

Antibiotic A 40926 factors PA and PB, at least under certainfermentation conditions, are the main antibiotic products of the A 40926producing microorganism.

Antibiotic A 40926 factors A and B are mainly transformation products ofantibiotics A 40926 factor PA and factor PB, respectively, and are oftenalready present in the fermentation broths.

It has been found that antibiotic A 40926 factor PA can be transformedinto antibiotic A 40926 factor A and antibiotic A 40926 factor PB can betransformed into antibiotic A 40926 factor B under basic conditionswhich lead to the removal of the acetyl group of the mannose unitwithout displacing the acyl group on the aminoglucuronyl unit.

As a consequence, when the fermentation broth or an antibiotic A 40926containing extract or concentrate thereof, is allowed to stand for acertain time under basic conditions (e.g. aqueous solution of anucleophilic base, at a pH>9 overnight) an antibiotic A 40926 complex isobtained which is enriched in antibiotics A 40926 factor A and factor B.

During the usual purification procedures of antibiotic A 40926 complex,factors PA and PB are largely converted to factors A and B.

Antibiotic A 40926 factor B can be obtained from A 40926 complex bychromatographic separation using the method described in U.S. Pat. No.4,935,238. Pure factor B₀ which under the conditions described in theabove mentioned European Patent account for about 90% of factor B, canbe obtained by further purification of factor B, for instance, byrepeated reverse-phase chromatography procedures.

More recent studies (L. Zerilli et al., Rapid Communications in MassSpectrometry, Vol. 6, 109, 1992) have shown that in the antibioticcomplex A 40926 also some minor factors are present, which have beenidentified with the acronyms A₁, RS-1, RS-2 and RS-3. These minorfactors have been individuated by HPLC and their structures have beendetermined by applying gas chromatography/mass spectrometry analysis ofthe methanolysates of the A-40926 complex.

All the above mentioned minor factors have structures corresponding tothe basic structure of factor A, B₀ and B₁ apart from the fatty acidresidues linked to the aminoglucuronic moiety. More particularly, makingreference to the formula (II), R has the same meanings as above while R₁represents: 8-methylnonyl in factor A₁, 7-methyloctyl in factor RS-1,n-nonyl in factor RS-2 and n-dodecyl in factor RS-3.

Although in the preparations of antibiotic A 40926 complex currentlycarried out under the fermentation conditions described in U.S. Pat. No.4,935,238 the factors wherein R₁ is a (C₁₀-C₁₁)alkyl are largelypredominant, it is possible to modify the fermentation conditions toincrease the amounts of the minor components wherein R₁ is a C₉ or a C₁₂alkyl.

All the sugar moieties are linked to the antibiotic A 40926 nucleusthrough O-glycosidic bonds.

In addition, it has been found that it is possible to transformantibiotic A 40926 complex, its single factors or a mixture of saidfactors in any proportion into the corresponding de-mannosyl derivatives(i.e. N-acylaminoglucuronyl aglycone complex AB, N-acylaminoglucuronylaglycone factor A, N-acylaminoglucuronyl aglycone factor B) bycontrolled acid hydrolysis of the mannosyl sugar moiety of the startingmaterial (see U.S. Pat. No. 4,868,171).

Preferred hydrolysis conditions for the production ofN-acylaminoglucuronyl aglycones comprise the usage of a mixture ofdimethylsulfoxide/concentrated hydrochloric acid from 8:2 to 9.5:0.5 ata temperature between 40° C. and 80° C.

Antibiotic A 40926 N-acylaminoglucuronyl aglycones are represented bythe above formula (II) wherein M is hydrogen, R is carboxy and R₁ is(C₉-C₁₂)alkyl.

Antibiotic A 40926 complex, the factors thereof, the correspondingN-acylaminoglucuronyl aglycones and mixtures thereof in any proportionare mainly active against gram positive bacteria and Neisseriae.

For the purposes of the present invention, each one of the above factorsor hydrolytic derivatives of antibiotic A 40926 may be employed asstarting materials for the present amidation process, either as a singlesubstance or as a mixture of two or more of them in any proportion. Theterm “mixtures” refers to the A 40926 complex obtained from a standardor modified fermentation process as known in the art, to thedemannosylated A 40926 complex thereof, to a complex obtained byapplying particular conditions in the isolation/purification of the A40926 complex or demannosylated A 40926 complex or to mixtures obtainedby mixing in the appropriate proportion the single factors of the A40926 complex and/or the hydrolyzed factors thereof, previously isolatedby means of chromatographic separation procedures.

International Pat. Appl. Publ. No. WO 92/17495, (designating also U.S.),describes ester derivatives of antibiotic A 40926 (esterified at theposition 6^(B), that is the carboxy group present on the N-acylaminoglucuronyl moiety) and its de-mannosyl derivatives are described; e.g.the compounds of formula (II) wherein R is (C₁-C₄)alkoxycarbonyl and R.and M have the same meanings of the symbols R₁ and M of formula I.

These ester derivatives are prepared by reacting the N¹⁵-protected (inthis description the term “N¹⁵” refers to the nitrogen atom of the aminofunction linked to the carbon atom at the 15-position of A 40926molecule) or N¹⁵-free amino A 40926 substrate or its demannosylderivative with an alkanol in an acid medium, or a N¹⁵-protected A 40926derivative or its demannosyl analogue with an alkyl halide (preferablybromide, chloride or iodide), optionally, in the presence of anhydrohalic acid acceptor, in particular, with an excess of the selectedalkanol in the presence of concentrated mineral acid at a temperaturebetween 0° C. and room temperature.

The above ester derivatives of antibiotic A 40926 obtainable from the A40926 starting material as above specified (single factors or mixturesthereof) are employed as intermediate compounds in the process disclosedin the International Patent Appl. Publ. No. WO 93/03060 (designatingalso U.S.) for preparing amide derivatives of antibiotic A 40926; amongthe amide derivatives of A 40926 disclosed therein, WO 93/03060discloses also the amide derivatives of formula I, together with aspecific process for obtaining them.

The amidation process reported in WO 93/03060 for preparing thecompounds of formula I consists essentially of 5 steps, which can besummarized as follows:

a) Preparation of an ester derivative on the 6^(B)-carboxy function of A40926;

b) protection of the amine function at the N¹⁵-position;

c) reduction of the ester moiety at the 6^(B)-position;

d) deprotection of the amine function at the N¹⁵-position;

e) amidation reaction on the C⁶³-carboxy function of A 40926.

Alternatively, steps c) and d) may be carried out after step e), i.e.the reduction of the ester moiety and the deprotection of the aminefunction may be carried out after the amidation reaction. The amidationaccording to the process disclosed in WO 93/03060 is carried out eitherin the presence of a condensing agent or via formation of an activatedester on the C⁶³-carboxy group.

According to WO 93/03060, the so obtained amide derivative is thenrecovered and purified by reverse phase column chromatography onsilanized silica gel, eluting with acetonitrile/acetic acid mixtures.

In this description and claims, when it is not otherwise specified, theterm “alkyl”, either alone or in combination with other substituents,includes both straight and branched hydrocarbon groups; moreparticularly, the term “(C₁-C₄)alkyl” represents a straight or branchedaliphatic hydrocarbon chain of 1 to 4 carbon atoms such as methyl,ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 1,1-dimethylethyl,and 2-methylpropyl.

As used herein, the terms “alk₁”, “alk₂” and “alk₃” represent anindependent linear or branched bifunctional aliphatic chain of 2 to 10carbon atoms such as for example:

—CH₂—CH₂—,

—CH₂—CH₂—CH₂—,

—CH₂—CH₂—CH₂—CH₂—,

—CH₂—CH₂—CH₂—CH₂—CH₂—,

—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—,

—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—,

—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—,

—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—,

—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—,

The term “(C₂-C₄)alkylene moiety” as used herein represents a linear orbranched bifunctional aliphatic chain of 2 to 4 carbon atoms.Representative examples of said chains can be drawn from the above list.

The expression “(C₁-C₄)alkoxycarbonyl” includes both straight andbranched alkoxycarbonyl groups such as for instance methoxycarbonyl,ethoxycarbonyl, propyloxycarbonyl, isopropyloxycarbonyl, butoxycarbonyl,isobutoxycarbonyl, and tert-butoxycarbonyl.

Here below are given some representative examples of the amino group

—NR₃-alk₁-(NR₄-alk₂)_(p)-(NR₅-alk₃)_(q)-W

according to the above definition:

and the like.

When R₃ and R₄ (or R₄ and R₅) taken together represent a (C₂-C₄)alkylenemoiety connecting the two nitrogen atoms, the saturated heterocyclicmoiety formed in combination with the portions alk₁ (or alk₂) and thetwo adjacent nitrogen atoms is preferably a piperazino ring.

For example, when R₃ and R₄ (or R₄ and R₅) taken together represent a(C₂-C₄)alkylene moiety connecting the two nitrogen atoms or when, both pand q being zero, W taken together with the moiety —NR₃-alk₁- representspiperazino or 4-methylpiperazino, the amino group of formula:

—NR₃-alk₁-(NR₄-alk₂)_(p)-(NR₅-alk₃)_(q)-W

identifies the following groups:

As said above, the present invention provides an improved process forpreparing the compounds of formula I. Said process involves a lowernumber of reaction's steps with respect to the process disclosed in WO93/03060, and further improvements relating both to the amidation stepand to the purification of the crude product of formula I, forincreasing the total yield of the process.

In particular, it has been found that the protection of the amino groupat the 15-position (step b) can be avoided without negatively affectingthe process course, while steps a) and c) can conveniently be carriedout in a one-pot reaction.

Furthermore, it has been found that, when the amidation step is carriedout in the presence of a condensing agent, it is possible to improve thereaction's yields by controlling the reaction conditions; in particular,attention should be paid in setting the initial pH of the reactingmixture, while also the amount of condensing agent and the amount ofamine reactant may be adjusted for improve the reaction's yield andminimize the formation of side-products.

In addition, a particularly advantageous chromatographic purificationprocedure has been set up, which involves the adsorbtion of the compoundof formula I onto a polyamide resin and wherein only aqueous solutionsare employed for the preliminar washings of the resin and the elution ofthe product.

Thus, one aspect of the present invention refers to the preparation ofthe intermediate compound of formula III:

wherein R₁ and M are as defined in formula I, by reacting a compound offormula II

wherein R₁ and M are as above defined, with a (C₁-C₄)alkanol in thepresence of a concentrated mineral acid, using the same alkanol as thereaction solvent and submitting the obtained ester compound to areductive process by adding an alkali metal borohydride into the samereaction mixture.

A further aspect of the present invention refers to the amidationreaction for obtaining the compound of formula I; this is carried out byreacting a compound of formula III with a suitable amine of formula IV

NHR₃-alk₁-(NR₄-alk₂)_(p)-(NR₅-alk₃)_(q)-W  IV

wherein R₃, R₄, R₅, alk₁, alk₂, alk₃, p, q and W are as defined informula I, in an inert organic solvent, in the presence of a condensingagent and setting the initial pH of the mixture (measured after dilutinga sample of the reaction mixture with 9 volumes of water) at a value offrom 6.5 to 9.0.

Furthermore, by combining the above improved steps into a singleprocess, it is possible to set up a new particularly convenient processfor preparing the compounds of formula I, which comprises:

a) reacting a compound of formula II with a (C₁-C₄)alkanol in thepresence of a concentrated mineral acid, using the same alkanol as thereaction solvent;

b) submitting the obtained compound to a reductive process with analkali metal borohydride, in the same reaction mixture, thus obtaining acompound of formula III;

c) reacting a compound of formula III with a suitable amine of formula

NHR₃-alk₁-(NR₄-alk₂)_(p)-(NR₅-alk₃)_(q)-W

 in an inert organic solvent, in the presence of a condensing agent andsetting the initial pH of the mixture (measured after diluting a sampleof the reaction mixture with 9 volumes of water) at a value of from 6.5to 9.0.

The reaction of a compound of formula II with a (C₁-C₄)alkanol can becarried out by following the process disclosed in WO 93/03060.Accordingly, the A 40926 starting material is contacted with an excessof the seleted (C₁-C₄)alkanol in the presence of a concentrated mineralacid at a temperature of from 0° C. to room temperature, for a timevarying from 4 to 24 hours.

The (C₁-C₄)alkanol is selected from methanol, ethanol, propanol andbutanol; preferably, ethanol is employed. As the alkanol acts both asreactant and as solvent, an excess of it is employed for theesterification reaction. Its molar amount may vary from 300 and 3000times the amount of the A 40926 starting material; preferably an excessof from 500 to 1500 moles is employed, particularly preferred being anexcess of about 650 moles.

Concentrated mineral acids are those known in the art, such asconcentrated sulfuric or hydrochloric acid. The acid is preferably addedto an alkanolic solution of the A 40926 starting material as a solutionwith the same alkanol. In general, the final concentration of the acidin the alkanolic reaction mixture will vary from 5 to 10% (w/v),preferably being about 7%, while the molar amount of acid will vary from15 to 50 times the molar amount of the A 40926 starting material,preferably about 20 times.

The alkanolic solution is generally cooled at about 0° C. before theaddition of the mineral acid, and the temperature is preferably kept atabout 0°-5° C. for a short time after the addition of the acid; then thetemperature is preferably allowed to rise during the esterificationreaction at about 20° C., while stirring the reaction mixture.

The reaction course is monitored by means of the conventional analyticaltechniques, for determining when the esterification reaction iscompleted; for this purpose, HPLC analysis is preferably employed. Thereaction time will depend from the temperature, the amount of alkanoland the concentration of the acid; for instance, when the molar amountof alkanol is about 650 times the amount of A 40926 starting material,the molar amount of acid is about 20 times, and the temperature is about20° C., the reaction may generally considered completed after about 18hours from the addition of the acid to the alkanolic solution of the A40926 starting material.

When the esterification reaction is completed, before the reduction stepwith alkali metal borohydride, the pH of the mixture is preferablyadjusted at a value of about 4.5-7.0 by addition of an aqueous alkalinesolution, preferably after having cooled the reaction mixture down toabout 0° C. to 5° C. Suitable solutions for adjusting the pH are aqueouscarbonate, bicarbonate or hydroxide solutions, in particular aqueousNa₂CO₃, NaHCO₃ or NaOH solutions; preferably an aqueous solution ofNa₂CO₃ is employed.

The so obtained hydro-alcoholic mixture is then treated with an alkalimetal borohydride, for reducing the ester group at position 6^(B) of theA 40926 molecule. The so obtained hydro-alchoholic mixture is thentreated with an alkali metal borohydride, reducing the ester group atposition 6^(B) of the A 40926 molecule, and thus obtaining the compoundof formula III.

Examples of suitable alkali metal borohydride are lithium borohydride,sodium borohydride, magnesium borohydride, sodium trimethossiborohydrideor sodium cyanoborohydride; particularly preferred is sodiumborohydride.

The amount of the alkali metal borohydride will vary from 5 or 8 to 50times the molar amount of the A 40926 ester derivative. Preferably anexcess from 8 or 10 to 16 times is employed, particularly preferredbeing an excess of 12 times.

The reaction course is monitored by means of the conventional analyticaltechniques, for determining when the reduction is completed; also inthis case, HPLC analysis is preferably employed. The reaction isgenerally completed in about 1 to 6 hours, depending on the temperatureand the excess of reducing agent.

When the reaction is completed, the excess of reducing agent isneutralized (for instance by addition of acetone) and the mixture istreated according to the known techniques for obtaining the crudecompound of formula III.

The amidation reaction is then carried out by reacting the compound offormula III with the desired amine of formula IV, in an inert organicsolvent, in the presence of a condensing agent. As said above, acritical reaction's parameter is the pH of the mixture, which should beadjusted at the beginning at a value of about 6.5-9.0 (measured afterdiluting the reaction mixture with 9 volumes of water), preferably fromabout 7.5 to 8.2.

The amine is preferably reacted in excess with respect to theintermediate compound of formula III, preferably from 1.6 to 2.2 molesper mole of intermediate, particularly preferred being about 1.8 molesper mole of intermediate.

For carrying out the amidation in the presence of a condensing agent, itis necessary that the amine reactant be capable of forming a salt withthe 63-carboxy function of the compound of formula III. In case theamine is not strong enough to form such a salt in the selected reactionmedium, it is necessary to add a salt-forming base (e.g. a tertiaryaliphatic or heterocyclic amine, such as triethylamine (TEA),N-methylpyrrolidine or N-methyl-piperazine, which cannot form an amidebond with the carboxy function) to the reaction mixture in an at leastequimolecular amount with respect to the A 40926 compound; a preferredsalt-forming base is TEA.

Use of a low molar excess of the amine reactant with addition of asalt-forming base is a suitable method when the amine reactant is arather expensive or difficult to obtain product.

It should be pointed out that, in general, the excess of amine added tothe reaction mixture will normally keep the pH above the suitable value;thus, for adjusting the pH at the desired value, a mineral acidpreferably diluted in an inert organic solvent can conveniently be addedto the mixture. Examples of suitable mineral acids are hydrochloric,sulfuric and phosphoric acid, while examples of inert organic solventsare dimethylformamide, dimethylsufoxide and dimethoxyethane; forinstance a solution of 27% hydrogen chloride in dimethylformamide ordimethylsulfoxide can suitably be employed.

Alternatively, the amine reactant may also be conveniently introduced inthe reaction medium as a corresponding acid addition salt, e.g. thehydrochloride. In this case a molar excess of a strong base capable offreeing the amine from its salts is added; the added excess should besuch to allow the initial pH of the mixture to be in the above range6.5-9.0. Also in this case, the suitable base is usually a tertiaryorganic aliphatic or heterocyclic amine which cannot form an amide bondwith carboxy functions like those exemplified above, preferably TEA. Infact, at least in some instances, the use of a salt of the amine whichis then freed in situ with the above mentioned bases, is highlypreferred, especially when the salt is more stable than thecorresponding free amine.

Suitable inert organic solvents are those organic aprotic solvents whichdo not unfavourably interfere with the reaction course and are capableof at least partially solubilizing the starting material.

Examples of said inert organic solvents are organic amides, ethers ofglycols and polyols, phosphoramides and sulfoxides. Preferred examplesof inert organic solvents are: dimethylformamide (DMF), dimethoxyethane,hexamethylphosphoramide, dimethylsulfoxide (DMSO) and mixtures thereof.Preferably, DMSO is employed.

The condensing agent employed in the amidation process of the inventionis one suitable for forming amide bonds in organic compounds and inparticular in peptide synthesis.

Representative examples of condensing agents are diisopropylcarbodiimide(DIC), dicylcohexylcarbodiimide (DCC) in the presence ofhydroxybenzotriazole (HBT),benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate,benzotriazolyloxy-tris-(pyrrolidino)phosphonium hexafluorophosphate and(C₁-C₄)alkyl, phenyl or heterocyclic phosphorazidates such as diphenylphosphorazidate, diethyl phosphorazidate,di-(4-nitrophenyl)phosphorazidate, dimorpholylphosphorazidate anddiphenylphosphoro-chloridate. The preferred condensing agents arediphenyl phosphorazidate, i.e. phosphoric acid diphenyl ester azide(DPPA), benzotriazolyloxy-tris-(dimethylamino)phosphoniumhexafluorophosphate (BOP), andbenzotriazolyloxy-tris-(pyrrolidino)phosphonium hexafluorophosphate(PyBOP).

Between the two last mentioned condensing agents PyBOP is particularlypreferred since the resulting by-product pyrrolidine has less potentialtoxicity problems than dimethylamine.

The amount of condensing agent may vary from 1 to 1.8 moles per mole ofcompound III, preferably being about 1.4 moles.

The reaction temperature will vary considerably depending on thespecific starting materials and reaction conditions.

In general, it is preferred to conduct the reaction at a temperaturebetween 0-30° C., preferably at about 5° C.

Also the reaction time will vary considerably depending on thecondensing agent and the other reaction parameters, such as temperature,molar amount of the reacting amine and steric complexity of it. Ingeneral, the condensation reaction is completed within a period of timefrom about one hour to about 24-48 hours.

In any case, the reaction course is monitored by TLC or, preferably, byHPLC according to methods known in the art.

On the basis of the results of these assays a man skilled in the artwill be able to evaluate the reaction course and decide when to stop thereaction and start working up the reaction mass.

The compound of formula I is then recovered as a crude product accordingto known per se techniques which include, for instance, extraction withsolvents, precipitation by addition of non-solvents, etc.. Preferably,the crude product is recovered by precipitation from the reactionmixture, for instance by addition of a non-solvent such as acetone,ethylacetate and the like, followed by filtration of the crudeprecipitate.

As previously stated, the present invention further provides an improvedpurification process for purifying the crude amide derivative of formulaI obtainable according to the above amidation process. Said purificationprocess comprises:

a) dissolving the crude product of formula I into an aqueous acidbuffered solution;

b) adsorbing said compound onto a polyamide resin;

c) washing the resin with the above aqueous acid buffered solution andthen with an aqueous basic solution;

d) eluting the compound with an aqueous acid solution and collectingthose fractions containing the purified compound of formula I.

The aqueous acid buffered solution referred to in step a) should be suchas to completely solubilize the crude compound of formula I, while thebuffer's constituents, apart from their solubilizing effect, shall not(or, in any case, only reversibly) interact with the compound of formulaI. Furthermore, as the same buffer is employed in step c) for the firstresin's washing, it should be such as to not (or only minimally) elutethe desired compound during the washing. Accordingly, preferred aqueousacid buffered solutions are those having a pH value from about 3.6 toabout 4.2, preferably from 3.8 to 4.0, particularly preferred being abuffered solution having a pH value of about 3.9. Suitable buffers aresolutions of organic acids with their respective alkali metal salts,such as acetate/acetic acid, formiate/formic acid; preferably, a sodiumacetetate/acetic acid buffer is employed.

The aqueous basic solution referred to in step c) should be such as toremove basic impurities adsorbed on the resin. Preferred solutions arethose having a pH value from about 8.5 to 9.2, preferably from 8.8 to9.0, particularly preferred being solutions having a pH value of about8.9. Suitable basic solutions are alkali metal salts of organic acidssuch as acetate or formiate; preferably sodium acetate is employed.

The aqueous acid solution referred to in step d) should be such as tocompletely elute the compound of formula I adsorbed on the resin.Preferred solutions are those having a pH value from about 3.2 to 3.6,preferably from 3.3 to 3.5, particularly preferred being solutionshaving a pH value of about 3.4. Suitable aqueous acid solutions may beprepared with organic acids such as acetic or formic acid; preferablyacetic acid is employed.

Polyamide resins that have been found useful in the present purificationprocess are selected from the polyamide column chromatography resinsgenerally identified as polycaprolactame, nylons (6/6, 6/9, 6/10 and6/12) and the cross-linked polyvinylpyrrolidone. Said chromatographypolyamide resins are generally characterized by a pore volume rangingbetween 1 and 5 ml/g, surface area(*) ranging between 1 and 100 m²/g,apparent density ranging between 0.15 and 0.50 g/ml, average porediameter(*) ranging between 100 and 3000 Å and particles sizedistribution where at least 40 percent of the particles have size lowerthan 300 micron (*=measured with a mercury porosimeter model Serie 200of C. Erba S.p.A., Milano Italy). Specific examples of commerciallyavailable polyamide column chromatography resins suitable for theembodiment of this invention are the polyamide resins Polyamide-CC 6,Polyamide-SC 6, Polyamide-CC 6.6, Polyamide-CC 6AC and Polyamide-SC 6ACof Macherey-Nagel & Co. (Germany), the polyvinylpyrrolidone resin PVP-CLof Aldrich Chemie Gmbh & Co., KG (Germany), the polyamide resin PA 400of M. Woelm (Germany). Particularly suitable is the resin polyamide-SC 6(Macherey-Nagel).

Although the above outlined purification process would be suitable forthe purification of any crude compound of formula I, independently fromwhether it is obtained according to the amidation process of theinvention or not, in view of the general improvements provided with thepresent invention, the skilled man will appreciate applying saidpurification process for preferably purifying the crude compounds offormula I obtained according to the improved amidation process disclosedabove.

The so obtained compound would generally have a purity degree suitablefor pharmaceutical use. Known per se recovery procedures may further beapplied, such as ultrafiltration, together with further treatments ofthe product, for instance depyrogenation when an injectable product isdesired.

Among the compounds defined by formula I, a group of preferred compoundswhich can be prepared according to the process of the present inventionare those compounds of formula I wherein R₁ represents (C₁₀-C₁₁)alkyl, Mrepresents α-D-mannopyranosyl and Y is as defined in formula I.

Within the above group, particularly preferred are those compoundswherein Y represents an amino group of formula:

—NR₂-alk₁-(NR₃-alk₂)_(p)-(NR₄-alk₃)_(q)-W

wherein:

R₂, R₃ and R₄ each independently represents hydrogen or (C₁-C₄)alkyl;

alk₁, alk₂

and alk₃ each independently represent a linear or branched alkylene of 2to 10 carbon atoms;

p and q are integers which independently represent zero or 1;

W represents hydrogen, (C₁-C₄)alkyl, amino, (C₁-C₄)alkylamino,di(C₁-C₄)alkylamino, amino substituted with one or twoamino(C₂-C₄)alkylene moieties or with one or two(C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties or with one or twodi(C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties, or, when both p and q arezero, taken together with the moiety —NR₃-alk₁- it may also representpiperazino or 4-methylpiperazino.

A further preferred group of compounds of formula I which may beprepared according to the process of the present invention is defined bythose compounds of formula I wherein: R₁ represents (C₁₀-C₁₁)alkyl, Mrepresents α-D-mannopyranosyl and Y represents an amino group offormula:

—NH-alk₁-(NH-alk₂)_(p)-(NH-alk₃)_(q)-W

wherein:

alk₁, alk₂

and alk₃ each independently represent a linear or branched alkylene of 2to 10 carbon atoms;

p and q are integers which independently represent zero or 1;

W represents hydrogen, (C₁-C₄)alkyl, amino, (C₁-C₄)alkylamino,di(C₁-C₄)alkylamino, amino substituted with one or twoamino(C₂-C₄)alkylene moieties or with one or two(C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties or with one or twodi(C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties.

The following examples are given to illustrate more in detail theimproved process of the present invention.

HPLC analyses were performed using a HP 1090 M instrument equipped witha DAD system connected to a work station. All chromatograms wererecorded at 254 nm following injection of 10 ml of solution. Inparticular:

a) for monitoring the esterification and reduction steps: BECKMANUltrasphere ODS 5 mm, 4.6×250 mm column at 40° C. with a flow rate of1.5 ml/minute; linear gradient from 28% to 58% of acetonitrile (30minutes) in buffered 0.02M NaH₂PO₄ (pH 6.5) as mobile phase;

b) for monitoring the amidation step: Asahipach ODP 50 5 μm, 4.6×250 mmcolumn at 40° C. with a flow rate of 0.9 ml/minute; linear gradient from26% to 56% of acetonitrile (25 minutes) in NaH₂PO₄ 0.05 M buffered (pH3.2) as mobile phase;

c) for analysis of the purified product: Asahipach ODP 50 5 μm, 4.6×250mm column and a Brownlee RP-18 7 μm. 3.2×15 mm pre-column at 40° C. witha flow rate of 0.9 ml/minute; acetonitrile (phase B) in NaH₂PO₄ 0.05 Mbuffered (pH 3.2) (35 min) as mobile phase, with the following gradient:

Time(min) 0 11 18 20 25 30 35 % B 26 33 33 40 43.5 54 26.

The titre reported for the A 40926 complex (starting material orderivatives thereof) refers to the percentage (w/w) of factors B₀+B₁ (orcorresponding derivatives thereof) with respect to the total amount ofdry sample.

EXAMPLE 1

Preparation of the intermediate compound of formula III wherein R₁ is(C₉-C₁₂)alkyl and M is α-D-mannopyranosyl

5 Kg of A 40926 complex obtained according to U.S. Pat. No. 4,935,238(HPLC titre=65.4%, corresponding to about 1.88 moles) and 37.5 l ofabsolute ethyl alcohol are loaded under stirring at room temperature ina 140 l glass lined reactor. The resulting suspension is cooled to 0° C.and then a solution containing 2 l of sulfuric acid in 12 l of absoluteethanol is added in 30 minutes maintaining the internal temperaturebetween 0-5 ° C.

The temperature is then left to rise at 20° C. while stirring iscontinued for an additional 18 hours. After this time the mixture iscooled again at 0-5° C. and adjusted to pH 5.8 by slowly adding 35 l ofa 10% aqueous sodium carbonate solution.

The mixture is then transferred into a 230 l stainless steel reactor and850 g of sodium borohydride (22.4 moles) dissolved in 9 l of water isslowly added, under stirring, with a peristaltic pump.

Stirring is continued at 5° C. for an additional two hours while thereduction is monitored by HPLC each hour by diluting a sample with 50parts of water.

The excess of reducer is completely destroyed with 3 l of acetone andthe resulting mixture is adjusted to pH 4.2 with 30% H₂SO₄. Thesuspension is then concentrated under vacuum at 40° C. to remove theorganic solvents and the residue is diluted with 50 l of water andfiltered under pressure (nitrogen 1.5 bar) on a stainless steel filter.The solid product is washed with 50 l of water and dried in a screwdryer under vacuum at 40° C. for 48 hours obtaining the title compound.

The above procedure is repeated on the same amount of A40926 startingmaterial (5 Kg, titre 65.4%), using the same reactor.

A total amount of 8.91 Kg of the crude title compound is obtained (titreabout 60%) with a molar yield of 82.2%.

EXAMPLE 2

Preparation of the 63-(dimethylaminopropyl)amido derivative ofantibiotic A 40926 (compound of formula I wherein Y is the group—NH—(CH₂)₃—N(CH₃)₂, R₁ is (C₉-C₁₂)alkyl and M is α-D-mannopyranosyl)

4.2 Kg of the crude compound obtained according to Example 1, 21.6 l ofDMSO and 5.4 l of DMF are loaded in a 140 l glass lined reactor and themixture is stirred until complete solubilization (90 minutes). Then 425ml of dimethylaminopropilamine are added in 10 minutes and its pH,measured after diluting a sample 9:1 with water, is adjusted to 8 byadding 310 ml of a previously prepared 27% HCl(g)/DMF solution.

The mixture is cooled at 5° C. and then a solution, prepared bydissolving 1.12 Kg of PyBOP in 4.5 l of DMF, is added in 20 minutes atroom temperature.

Stirring is continued for an additional hour, then the mixture istransferred in a 700 l glass lined reactor where the final product isprecipitated with 150 l of ethyl acetate.

The suspension is filtered on a stainless steel filter and the solidobtained is washed on the filter with 30 l of ethyl acetate and dried ina stainless steel screw drier at 35° C. under vacuum for 24 hours,obtaining the title compound as a crude.

The above procedure is repeated on the same amount of A40926 startingmaterial, using the same reactor.

A total amount of 10.4 Kg of the crude title compound is thus obtained(titre about 38%) with a molar yield of 76.0%.

EXAMPLE 3

Chromatographic purification of the 63-(dimethylaminopropyl)amidoderivative of antibiotic A 40926 obtained according to Example 2.

900 g of the above crude material are dissolved in acetate buffer pH 3.9(about 30 l ) and applied on the top of a chromatographic column (ID=30cm), filled with about 56.5 l of polyamide resin (SC-6, Machery-Nagel)previously equilibrated with about 180 of the same acetate buffer.

The column is washed with about 120 l of the same acetate buffer andthen with about 400 l of a 0.1 M sodium acetate solution (pH=8.9).

The compound of formula I is then eluted with about 400 l of acetic acid0.1 M (pH 3.4), monitoring each fraction by HPLC and collecting thosefractions containing the title compound (chromatographic yield about80%).

What is claimed is:
 1. Process for preparing a compound of formula I,

wherein: R₁ represents (C₉-C₁₂)alkyl; M represents hydrogen,α-D-mannopyranosyl or 6-O-acetyl-α-D-mannopyranosyl; Y represents anamino group of formula —NR₂-alk₁-(NR₃-alk₂)_(p)-(NR₄-alk₃)_(q)-Wwherein: R₂ represents hydrogen or (C₁-C₄)alkyl; alk₁, alk₂ and alk₃each independently represent a linear or branched alkylene of 2 to 10carbon atoms; p and q are integers which independently represent zero or1; R₃ and R₄ each independently represent hydrogen, (C₁-C₄)alkyl or R₂and R₃ taken together represent a (C₂-C₄)alkylene moiety connecting thetwo nitrogen atoms with the proviso that p is 1; or R₃ and R₄ takentogether represent a (C₂-C₄)alkylene moiety connecting the two nitrogenatoms with the proviso that both p and q are 1; W represents hydrogen,(C₁-C₄)alkyl, amino, (C₁-C₄)alkylamino, di(C₁-C₄) alkylamino, aminosubstituted with one or two amino-(C₂-C₄)alkylene moieties or with oneor two (C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties or with one or twodi(C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties, or, when both p and q arezero, taken together with the moiety —NR₂-alk₁- it also representspiperazino or 4-methylpiperazino, which comprises reacting a compound offormula III

wherein R₁ and M are as defined in formula I, with a suitable amine offormula IV NHR₂-alk₁-(NR₃-alk₂)_(p)-(NR₄-alk₃)_(q)-W  IV wherein R₂, R₃,R₄, alk₁, alk₂, alk₃, p, q and W are as defined in formula I, in aninert organic solvent, in the presence of a condensing agent,characterized in that the initial pH of the mixture, measured afterdiluting a sample of the reaction mixture with 9 volumes of water, isset at about 7.5-8.2.
 2. Process according to claim 1 wherein a compoundof formula III

wherein R₁ is (C₉-C₁₂)alkyl and M is hydrogen, α-D-mannopyranosyl or6-O-acetyl-α-D-mannopyranosyl, is prepared by a process which comprisesreacting a compound of formula II

wherein R₁ and M are as above defined, with a (C₁-C₄)alkanol in thepresence of concentrated mineral acid, using the same alkanol as areaction solvent and submitting the obtained esterified compound to areductive process by adding an alkali metal borohydride into the samereaction mixture.
 3. Process according to claim 2 wherein the(C₁-C₄)alkanol is ethanol.
 4. Process according to claim 2 wherein theamount of alkanol is from 300 to 3000 times and the amount of mineralacid is from 15 to 50 times the molar amount of the compound of formulaII.
 5. Process according to claim 2 wherein the amount of alkanol is 650times and the amount of mineral acid is about 20 times the molar amountof the compound of formula II.
 6. Process according to claim 2 whereinthe pH of the reaction mixture is adjusted to a value of about 4.5-7.0before the reduction with alkali metal borohydride.
 7. Process accordingto claim 2 wherein the alkali metal borohydride is sodium borohydride.8. Process according to claim 2 wherein the amount of alkali metalborohydride is from 8 to 50 times the molar amount of the esterifiedcompound.
 9. Process according to claim 2 wherein the amount of alkalimetal borohydride is about 12 times the molar amount of the esterifiedcompound.
 10. Process according to claim 1 wherein the amount of amineis from 1.6 to 2.2 times the molar amount of the compound of formulaIII.
 11. Process according to claim 1 wherein the amount of amine isabout 1.8 times the molar amount of the compound of formula III. 12.Process according to claim 1 wherein the condensing agent is selectedfrom diisopropylcarbodiimide or dicyclohexylcarbodiimide in the presenceof hydroxybenzotriazole;benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate;-benzotriazolyloxy-tris-(pyrrolidino)-phosphonium hexafluorophosphate;and (C₁-C₄)alkyl, phenyl or heterocyclic phosphorazidates.
 13. Processaccording to claim 1 wherein the condensing agent isbenzotriazolyloxy-tris-(pyrrolidino)phosphonium hexafluorophosphate. 14.Process according to claim 12 wherein the amount of condensing agent isfrom 1 to 1.8 moles per mole of compound of formula III.
 15. Processaccording to claim 12 wherein the amount of condensing agent is about1.4 moles per mole of the compound of formula III.
 16. Process accordingto claim 1 for preparing a compound of formula I wherein R₁ represents(C₁₀-C₁₁)alkyl, M represents α-D-mannopyranosyl and Y is as defined inclaim
 1. 17. Process according to claim 16 wherein Y represents an aminogroup of formula: NR₂-alk₁-(NR₃-alk₂)_(p)-(NR₄-alk₃)_(q)-W wherein: R₂,R₃ and R₄ each independently represents hydrogen or (C₁-C₄)alkyl; alk₁,alk₂ and alk₃ each independently represent a linear or branched alkyleneof 2 to 10 carbon atoms; p and q are integers which independentlyrepresent zero or 1; W represents hydrogen, (C₁-C₄)alkyl, amino,(C₂-C₄)alkylamino, di(C₁-C₄)alkylamino, amino substituted with one ortwo amino (C₂-C₄)alkylene moieties or with one or two(C₁-C₄)alkylamino-(C₂C₄)alkylene moieties or with one or twodi(C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties, or, when both p and q arezero, taken together with the moiety —NR₂-alk₁- it also representspiperazino or 4-methylpiperazino.
 18. Process according to claim 16wherein Y represents an amino group of formula:—NH-alk₁-(NH-alk₂)_(p)-(NH-alk₃)_(q)-W wherein: alk₁, alk₂ and alk₃ eachindependently represent a linear or brached alkylene of 2 to 10 carbonatoms; p and q are integers which independently represent zero or 1; Wrepresents hydrogen, (C₁-C₄)alkyl, amino, (C₁-C₄)alkylamino,di(C₁-C₄)alkylamino, amino substituted with one or twoamino(C₂-C₄)alkylene moieties or with one or two(C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties or with one or twodi(C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties.
 19. Process for preparinga compound of formula I,

wherein: R₁represents (C₉-C₁₂)alkyl; M represents hydrogen,α-D-mannopyranosyl or 6-O-acetyl-α-D-mannopyranosyl; Y represents anamino group of formula —NR₂-alk₁-(NR₃-alk₂)_(p)-(NR₄-alk₃)_(q)-Wwherein: R₂ represents hydrogen or (C₁-C₄)alkyl; alk₁, alk₂ and alk₃each independently represent a linear or branched alkylene of 2 to 10carbon atoms; p and q are integers which independently represent zero or1; R₃ and R₄ each independently represent hydrogen, (C₁-C₄)alkyl or R₂and R₃ taken together represent a (C₂-C₄)alkylene moiety connecting thetwo nitrogen atoms with the proviso that p is 1; or R₃ and R₄ takentogether represent a (C₂-C₄)alkylene moiety connecting the two nitrogenatoms with the proviso that both p and q are 1; W represents hydrogen,(C₁-C₄)alkyl, amino, (C₁-C₄)alkylamino, di(C₁-C₄)alkylamino, aminosubstituted with one or two amino-(C₂-C₄)alkylene moieties or with oneor two (C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties or with one or twodi(C₁-C₄)alkylamino-(C₂-C₄)alkylene moieties, or, when both p and q arezero, taken together with the moiety —NR₂-alk₁- it also representspiperazino or 4-methylpiperazino, which comprises reacting a compound offormula III

wherein R₁ and M are as defined in formula I, with a suitable amine offormula IV NHR₂-alk₁-(NR₃-alk₂)_(p)-(NR₄-alk₃)_(q)-W  IV wherein R₂, R₃,R₄, alk₁, alk₂, alk₃, p, q and W are as defined in formula I, in aninert organic solvent, in the presence of a condensing agent,characterized in that the initial pH of the mixture, measured afterdiluting a sample of the reaction mixture with 9 volumes of water, isset at a value of from 6.5 to 9.0, and wherein the compound of formula Iis purified according to the following sequence of steps: a) dissolvingthe crude product of formula I into an aqueous acid buffered solution;b) adsorbing said compound onto a polyamide resin; c) washing, the resinwith the above aqueous acid buffered solution and then with an aqueousbasic solution; d) eluting the compound with an aqueous acid solutionand collecting those fractions containing the purified compound offormula I.
 20. Purification process according to claim 19 wherein thepolyamide resin is a polyamide column chromatography resin selected frompolycaprolactame, nylons 6/6, 6/9, 6/10 and 6/12, and cross-linkedpolyvinylpyrrolidone.
 21. Process according to claim 19 for purifying acompound of formula I wherein the aqueous acid buffered solutionreferred to in step a) and step c) has a pH value from about 3.6 toabout 4.2.
 22. Process according to claim 21 wherein the aqueous acidbuffered solution referred to in step a) and step c) has a pH value fromabout from 3.8 to 4.0.
 23. Process according to claim 21 wherein theaqueous acid buffered solution is an aqueous acetate/acetic acid oraqueous formate/formic acid buffered solution.
 24. Process according toclaim 19 for purifying a compound of formula I wherein the aqueous basicsolution referred to in step c) has a pH value from about 8.5 to 9.2.25. Process according to claim 24 wherein the aqueous basic solution hasa pH value from about 8.8 to 9.0.
 26. Process according to claim 24wherein the aqueous basic solution is an acetate or formate solution.27. Process according to claim 19 for purifying a compound of formula Iwherein the aqueous acid solution referred to in step d) has a pH valuefrom about 3.2 to 3.6.
 28. Process according to claim 27 wherein theaqueous acid solution has a pH value from about 3.3 to 3.5.
 29. Processaccording to claim 27 wherein the aqueous acid solution is an acetic orformic acid solution.
 30. Purification process according to claim 20wherein the polyamide resin is characterized by a pore volume rangingbetween 1 and 5 ml/g, surface area ranging between 1 and 100 m²/g,apparent density ranging between 0.15 and 0.50 g/ml, average porediameter ranging between 100 and 3000 Å (10 and 300 nanometer) andparticle size distribution where at least 40 percent of the particleshave a size lower than 300 micrometers.